This invention relates to optical scanning of substrates, and in particular to efficient and uniform collection of emitted or scattered light. The invention includes the application of phase sensitive detection to scanned images to improve the discrimination of multiple signal sources and reduce noise. It is particularly useful for automated, rapid and sensitive fluorescent gel scanning.
Large-scale genome and proteome projects involve assay techniques that are significantly hindered by current substrate imaging techniques. For example, gel electrophoresis is a critical but slow step in analyses of nucleic acid sequences and proteins. DNA or protein samples are often electrophoresed in a gel, which separates sample components, and this is followed by formation of an image for quantitation. A gel image can be produced by using a radio-labeled sample and exposing the gel to an x-ray film or storage phosphor plate followed by film development or phosphorescence quantitation. Radioactive labeling, while sensitive, is hazardous and expensive. Fluorescent dyes which bind to the sample and thereupon fluoresce brightly are a preferred method of sample labeling. Application of fluorescence labeling to slab gel electrophoresis and other two-dimensional analysis substrates creates a need for fluorescence imaging instrumentation. Fluorescent intensity images can be created by photographing the substrate or imaging it with a charge coupled device (CCD) areal detector chip. Photography is non-quantitative due to the film""s highly non-linear exposure/density function. A CCD detector is capable of quantifying the fluorescence intensity of an image but the spatial resolution is limited by the number of pixels on the chip itself. Both forms of detection involve the use of cameras which have relatively poor collection efficiency, rely on broad and even illumination, and require optics which minimize geometric distortion. These constraints become more acute when imaging large substrates such as electrophoresis gels. Further, cameras are not configured for the rejection of background light, resulting in high background levels.
One alternative for fluorescence quantitation of substrates is image scanning. The fluorescence is measured sequentially at each point in a substrate, creating an image based upon millions of individual pixel measurements. Scanning systems quantify only one point at a time and do not require imaging optics, allowing the optical system to be optimized for collection efficiency and to satisfy other constraints. Fluorescence is typically excited by a laser, which is far brighter and more uniform than other forms of illumination. There are two types of designs for scanning systems and in both types the time required to produce a scanned image increases with the area of the substrate. Scanning-head designs physically translate the excitation and collection optics over the substrate area, resulting in high collection efficiency and noise rejection at the expense of speed. Scanning-beam designs, also commonly termed scanning-spot and flying-spot, accomplish rapid movement of an illumination spot separate from a stationary or slow-moving collection system, imaging rapidly at the expense of collection efficiency.
The designs of existing fluorescence scanning devices provide a trade-off between scanning optic detection systems with high sensitivity and scanning beam detection systems with speed but low sensitivity. Neither system architecture achieves maximum image noise rejection because neither system architecture can eliminate non-random background noise. Background noise consists, in part, of excitation light scattered from the substrate itself as well as from surface and bulk contaminants. Scatter intensity varies with the characteristics of the substrate. Membranes are generally opaque and therefore scatter nearly all the excitation light. Agarose gels are translucent, scattering a fraction of the incident excitation light. Polyacrylamide gels are very clear and exhibit the least scatter. The scattered light component of background noise can consist of elastic scatter at the same wavelength as the incident light and Raman (inelastic) scatter that is red-shifted due to interaction between incident light and vibrating hydrogen-oxygen bonds of water in the substrate. Elastically-scattered light is largely blocked by an emission filter, but it can induce fluorescence in the filter itself which cannot be distinguished from signal fluorescence. Raman scatter often overlaps the emission spectrum of the fluorescent dye, thereby making it past the emission filter. Other significant components of background noise include fluorescence from unbound dyes and autofluorescence of the spectral filter and glass or plastic substrate materials. The foregoing components of background noise are considered to be non-random signals and cannot be removed by conventional signal-averaging techniques.
Therefore, there is a need in the art to provide a high-throughput image scanning device that produces high sensitivity and low background noise fluorescent images, thereby increasing the information content in an image for a given amount of nucleic acid or protein material. Ideally, the device would suppress undesired non-random signals to such a degree that faint fluorescence signals could be imaged even on opaque, scattering, or highly auto-fluorescent substrates.
The present invention provides a scanning apparatus and methods to obtain automated, rapid and sensitive scanning of substrate luminescence (fluorescence, phosphorescence, chemiluminescence, nano-particle emission, etc.), optical density or reflectance. The scanning apparatus employs moving-beam excitation to rapidly measure samples on or in a variety of substrates, including fluorescently-stained gels, silver-stained gels, developed x-ray films, storage phosphor screens, membranes, multi-well plates, petri dishes, glass and plastic surfaces, silicon chips and other emitting, reflecting or scattering substrates. The scanning apparatus employs a constant path length optical train to achieve highly uniform images with a minimum of optical complexity and no need for focus adjustment for a variety of substrates with widely-varying shapes, sizes, thicknesses, and optical characteristics. The constant optical path length also facilitates the use of phase sensitive signal processing. A method is provided for the use of phase nulling, either electronically or by a combination of electronics and software, to eliminate non-random baseline components and thereby enable the use of signal averaging to improve the signal-to-noise ratio. A method is further provided to allow the use of phase-sensitive detection for the improved discrimination of multiple dye fluorescence signals in the same substrate on the basis of their excited-state lifetimes.
The constant path length optical train of this invention is used to direct light onto and collect light from a substrate. It comprises a light source, a scanning mirror to receive light from the light source and sweep it across a steering mirror, a steering mirror to receive light from the scanning mirror and direct it to the substrate, whereby it is swept across the substrate along a scan arc, a waveguide or mirror to collect the optical signal, and a photodetector to receive emitted or scattered light from the substrate, wherein the optical pathlength from the light source to the photodetector is substantially constant throughout the sweep across the substrate.
The scan mirror preferably has one or more flat surfaces and is mounted on a scanning motor to rotate the mirror and thereby scan the reflected beam. Although the rotation can be oscillating, it is preferably unidirectional and at constant speed and therefore produces a constant angular sweep and constant incident optical power on the substrate per unit time. The scanning mirror can alternatively be curved, thereby simultaneously providing beam movement and focusing. In a first embodiment, the mirror has a single flat reflecting surface at the axis of rotation. Placement of a single reflecting surface at the axis of rotation prevents any path length variation arising from translation of the reflecting surface during rotation. For a 22.5xc2x0 physical sweep of a single reflecting surface, corresponding to a 45xc2x0 optical sweep employed in the first embodiment, the illumination duty cycle is only 1:16.
In a second embodiment the mirror is multifaceted, having a plurality of reflecting surfaces. The mirror can be a polygon centered at the axis of rotation. In a preferred embodiment, the polygon has 16 sides, each side providing a 22.5xc2x0 physical sweep. The polygonal mirror increases the duty cycle by a factor of 16. The number of sides can be selected in accordance with the sweep angle and data acquisition electronics of the particular embodiment to optimize the duty cycle. However, since each surface of the polygon is displaced from the axis of rotation, the path length varies over the course of the scan. This variation in path length is a small percentage of the total path length, about one millimeter out of over 300 mm in a tested embodiment. At the operating frequency of the phase sensitive electronics in the tested embodiment, the phase shift caused by this path length variation is below the detection limit of the scanner. In this system a 1xc2x0 phase shift is detectable, which corresponds to over 5 mm path length difference. The term xe2x80x9csubstantially constant path lengthxe2x80x9d refers to a path length wherein the variations produce a phase shift which is too small to interfere with signal detection and analysis, and more preferably is below the detection limit of the scanner. At higher operating frequencies, the phase variation caused by the faceted mirror may be detectable, but is deterministic, allowing it to be corrected by a software analysis of the signal.
The steering mirror is preferably curved with a radius of curvature equal to the distance between the scanning mirror and the steering mirror and preferably between about 2 times and about 5 times the width of the substrate, thereby producing a constant optical path length and a constant plane of focus. This gives the steering mirror a cylindrical curvature and produces a correspondingly curved scan arc. Since the steering mirror is preferably beveled at approximately 45xc2x0 to direct the light at normal incidence or beveled at approximately 73xc2x0 to direct the light at Brewster""s angle, the mirror is a conic section.
The optical train can further include a waveguide to collect emitted or scattered light from the substrate and direct it to the photodetector. To maintain the constant path length and uniform collection efficiency, the waveguide is preferably curved at the collection end to match the curvature of the scan arc. For a single photodetector, the waveguide is preferably wedge-shaped, tapering toward the photodetector end. The waveguide is preferably beveled at the collection end to couple light in and beveled at the detection end to reflect light into the photodetector. There is a range of internal reflection paths which can be followed to couple light from the substrate to the photodetector, with a corresponding a range of path lengths. However, the waveguide and photodetector dimensions are selected so that the path length is substantially constant.
The waveguide can be shaped to provide one-dimensional confocal imaging of the excitation spot. In conventional confocal imaging, crosstalk is minimized by forming an image of the excitation spot on a pinhole aperture placed between the substrate and the photodetector using imaging optics which move along with the excitation beam. Scatter from neighboring pixels is blocked by the pinhole. Confocal imaging increases spatial resolution and reduces scatter crosstalk at the cost of increased optical complexity and reduced scan speed. In the scanner, one dimensional confocal imaging can be achieved using a waveguide with a curved collection edge rather than a flat bevel. The edge profile is an off-axis parabolic section with a focal point coincident with the illumination spot. The reflection characteristic of a parabolic section causes rays emanating from the illumination spot to be reflected into the plane of the waveguide, parallel to one another. Rays from pixels located adjacent to the excitation spot are not transmitted in the plane of the waveguide. In lieu of or in addition to a 45xc2x0 bevel at the detector end, a cylindrical focuser, e.g., a curved waveguide edge, a lens or a mirror, is used to focus the rays into a line. The lens can be integral to or external to the waveguide. A slit is used to select only those rays in the line which originate from the illumination spot, and thereby block crosstalk. The detector aperture can function as the slit.
Confocal collection can also be achieved through the use of an off-axis parabolic collection mirror in place of the waveguide. To maintain the constant path length and uniform collection efficiency, the collection mirror is preferably curved to match the scan arc. Rays emanating from the illumination spot are reflected parallel to the plane of the scanner platform and are directed towards the detector. Rays from adjacent pixels are reflected out of the plane and either absorbed by the surrounding structure or the slit aperture, never reaching the detector. In the embodiment employing a parabolic collection mirror, there is no need for the waveguide since parallel rays originating from the illumination spot do not require internal reflection to reach the detector. The use of reflective collection optics rather than transmissive collection optics eliminates many restrictions on the wavelengths of light which can be employed in illumination and detection. For example, the use of a collection mirror allows the imaging of storage phosphor plates based on their 390 nm phosphorescence emission. The acrylic waveguide of the tested embodiment cannot be used in this application because the material absorbs all light below 400 nm wavelength. This signal would otherwise require a glass waveguide, which transmits wavelengths as short as 300 nm. Similarly, a scanner employing a collection mirror can be used to measure DNA concentration directly in gels via the characteristic DNA absorbance of 260 nm wavelength light. Even a glass waveguide would absorb this signal, requiring use of silica or another expensive and difficult to fabricate waveguide material.
Although it is preferred that both the path length from the light source to the substrate and from the substrate to the detector be constant, it is sufficient that the path length from the light source to the detector be constant. This can be accomplished with a flat steering mirror by configuring the collection optics, and in particular the waveguide, to compensate for path length differences introduced by the illumination optics.
The optical train can include additional beam shaping and directing elements. There can be a beam expander and a focusing mirror or lens. The optical connection between the light source, the scanning mirror, the steering mirror, the substrate and the detector can include mirrors which redirect the path without substantially changing the beam characteristics. There can be spectral filters between the detector and the waveguide or collection mirror.
This invention further comprises a scanner comprising the optical train in combination with a substrate holder. The scanning mirror provides scanning in one dimension. For substrates requiring two dimensional optical images, for example slab gels, the substrate holder can be a substrate translator. The substrate is translated in one dimension while the scanning mirror scans the beam in a second, preferably substantially orthogonal, dimension. The term xe2x80x9csubstantially orthogonalxe2x80x9d is used because the scan arc is curved. In the preferred embodiment the steering mirror is curved and the substrate is flat. In lieu of or in addition to the curve in the steering mirror, the substrate itself can be curved to provide a constant optical path length. For example, if two-dimensional scanning is accomplished using a substrate translator, the steering mirror can be flat and the substrate can be cylindrical. For two-dimensional scanning without a substrate translator, the steering mirror can be enlarged to allow beam translation over an area. In this case, a motor rotates the scan mirror in one axis while a second actuator pivots the entire scan motor/mirror assembly. With flat substrates, the steering mirror curvature must compensate for the path length change induced by the pivoting scan mirror assembly. Conversely, a flat steering mirror can be employed with a curved substrate.
For high throughput automated operation, the substrate translator is preferably a conveyor belt. This invention provides a high throughput scanner wherein substrates are loaded onto the conveyor belt from a tray feeder. The scanner can further include a sample preparation apparatus such as a sample loader, a gel stainer and integral electrophoresis apparatus. The scanner can further include an apparatus for extracting sample fractions from the gel, multi-well plate, petri dish, membrane, or other substrate. When the scanner detects a band of interest, the sample extractor can be moved into position to remove a sample by excising that section of the substrate or by freeing the sample from the substrate by physical, chemical, electrical, or other means.
Because the optical path has a constant length, phase sensitive detection (PSD) can be used to distinguish multiple luminescent sources with different excited-state lifetimes and/or minimize undesired signal components. The excited-state lifetime of a luminescent species introduces a time delay between the absorption of exciting light and the emission of fluorescence, phosphorescence, or other optical signal. This temporal information can be useful in distinguishing desired luminescence from scattered light and non-probe luminescence. In PSD, the light source intensity is modulated, preferably sinusoidally. The luminescent emission is, in turn, modulated at the same frequency. The time delay between excitation and emission creates a phase shift between scattered and emitted light. By the use of both spectral and temporal information, the discrimination of signal components is improved, increasing the signal to noise ratio of luminescence images and increasing the flexibility of multicolor systems.
This invention provides a PSD scanner comprising the constant path length optical train with an intensity modulated light source. The light source can be modulated internally, for example by varying the current supplied to a diode laser, or externally, for example by an acousto-optic modulator. It is preferred that the circular frequency of the modulation be the reciprocal of the dye lifetime, resulting in a phase shift of 45xc2x0 between the excitation and emission light. Modulation frequencies are typically between 1 and 100 MHz. The scanner also comprises phase-sensitive detection electronics to distinguish between the baseline signal and one or more luminescent signals based on the phase difference. The phase-sensitive detection electronics can comprise a frequency mixer which multiplies the signal input from the photodetector with a reference input. The reference input can be driven by the light source modulator, by a photodetector receiving a portion of the modulated light, or by a signal generator. The mixer is followed by a low-pass filter which demodulates the two inputs. Either the in-phase or quadrature channel of a lock-in amplifier can also provide these functions. The phase-sensitive detection electronics can also include additional signal processing elements such as amplifiers, filters and buffers.
In a preferred embodiment of the PSD scanner, phase nulling is used to reduce non-random components of the baseline signal arising from sources such as elastically scattered excitation light, Raman scattered light, fluorescence from unbound dye, and fluorescence from the emission filter and substrate. One embodiment of the phase-nulling system comprises a demodulator circuit or one leg of a quadrature lock-in amplifier, which functions as a demodulator, and further comprises a reference signal phase adjuster. The output of the demodulator is proportional to the cosine of the phase difference between the detector signal and the reference signal. The detector and reference signals are input to the demodulator circuit and the reference phase is adjusted so that it is 90xc2x0 out of phase with the baseline signal, thereby nulling it completely. The reference signal comes from an RF signal generator phase-locked to the light source modulator. The signal generator is used as a phase adjuster to add or subtract phase delay from the modulated excitation waveform. Before scanning the substrate for an image, the reference phase is calibrated, typically manually, to null the baseline signal in a region of the substrate known not to contain any signals of interest. Luminescence signal components in the substrate image which are out of phase with the baseline signal are only partly attenuated by the phase-nulling system. Minimum attenuation of the luminescence signal occurs when there is a 45xc2x0 phase shift between the luminescence and baseline components.
In a second embodiment of the phase-nulling system, phase nulling is accomplished mathematically in software. The phase-sensitive detection electronics utilize both the in-phase and quadrature demodulation legs of a lock-in amplifier. The reference source does not require a phase adjustor because of the quadrature splitting of the reference signal in the lock-in. Using a computer-implemented method for phase nulling, for which the relationships are provided herein, the phase shift of the luminescence signal is obtained from the in-phase and quadrature outputs of the phase-sensitive detection electronics. Phase nulling in software is particularly useful for substrates having more than one fluorescent dye or for which it is impractical to manually adjust the reference signal in the first embodiment.
Phase nulling removes the non-random baseline noise and leaves primarily random noise. While signal averaging is ineffective in reducing constant baseline noise, it can reduce the random noise which remains after phase nulling. The phase-nulling PSD scanner can further comprise a signal averager. The averager can operate in a variety of ways. In one method, the speed of the scanner is reduced so that multiple sequential measurements of each pixel are averaged over time. In a second method, the scanner makes multiple high-speed sweeps over each scan line in the substrate. The scans are accumulated and each pixel in the scan line is averaged by the number of passes. In a third method, the scanner traverses the entire substrate multiple times and accumulates the images. Each pixel value is then averaged by the number of images accumulated. In scanner embodiments employing multiple scan mirror facets, averaging speed can be increased due to the higher duty cycle of data acquisition.
In addition to reducing non-random noise in luminescence images, phase nulling can be employed to enhance discrimination between multiple luminescent sources in the same image. Multi-dye imaging is highly useful for such things as DNA sequencing, where a different fluorescent dye is used to label each of the four species of nucleotide and one or more additional dyes may be used to define an electrophoresis lane. In conventional DNA sequencing instruments, the multiple dyes are discriminated on the basis of their emission spectra. The wide spectral range encompassed by the dye set makes it difficult to efficiently excite all the dyes with a single laser, requiring multiple lasers per instrument or drastic dye concentration differences and/or expensive hybrid dyes which use resonant energy transfer to partially normalize excitation efficiency in single laser instruments. Regardless of the means of excitation, emission spectral overlap between the dyes gives rise to channel crosstalk, reducing discrimination. These problems can be avoided by employing multiple phase-nulling circuits, each tuned to null a single signal component. In the case of four-color DNA sequencing, four nulling circuits are used with four separate reference phase delays.
In one implementation, all four dyes have similar spectral characteristics but different excited state lifetimes. Each reference signal nulls the fluorescence from one dye. The identity of each signal peak is then determined by the presence of partially-attenuated signal peaks in three channels and complete nulling in the remaining channel. In a second implementation, the dyes are paired into two spectral classes that are comparably well-excited by a single laser. Within each spectral class, the dyes have different excited state lifetimes. Multiple phase-nulling circuits, one per excited state lifetime, are used as in the first implementation to separately null the dye signals. Multiple emission filters are used to discriminate dyes which have similar excited state lifetimes but different spectral classes. Both implementations can employ either the manual nulling method or the software-based nulling method, described above.
This invention provides a method of noise reduction in the detection of modulated luminescence comprising the steps of demodulating the detected signal using a phase-labile reference signal, phase nulling the detected signal to reduce non-random noise, and signal averaging to reduce random noise. The phase nulling can be performed electronically or in software, as described above.
This invention further provides a computer-implemented method and apparatus for phase nulling, comprising a lock-in amplifier to obtain the in-phase and quadrature demodulated outputs from a signal and a reference input of arbitrary phase, and a computer to obtain the phase via the relationships provided herein.
This invention further provides a method of signal discrimination for use with multiple sources of modulated luminescence, comprising the steps of phase nulling each luminescence signal source and determining the identity of a signal component based on the presence of signals in all but one phase nulling circuit.
This invention further provides a multiple image-type scanner. The scanners described above can be adapted for use with a variety of image types, including fluorescent gels, silver-stained gels, developed x-ray films, storage phosphor screens, membranes, multi-well plates, and other emitting, reflecting, transmitting or scattering substrates. The multiple image-type scanner has a plurality of light sources and/or a removable diffuse reflector. The type of light source, the spectral filters, and the use of a diffuse reflector are interchanged according to the image type. For measuring images on reflective substrates, for example a black and white photograph, light is absorbed in some portions and reflected in others. By detecting the reflected light, the image can be reconstructed. For measuring optical density of transmissive substrates, for example x-ray films, a diffuse reflector is positioned behind the substrate as a scatter source. At high optical density the light is absorbed by the film and not scattered back to the detector, while at low optical density it is transmitted through the substrate, scattered by the diffuse reflector, transmitted again through the substrate, and detected by the photodetector. For detection of emitted light the diffuse reflector is removed. For fluorescent images, the light source is preferably an argon ion laser. For phosphor screens, the light source is preferably a helium-neon laser.
The several aspects of this invention can be used separately or in combination to provide high throughput and high sensitivity analysis of substrates.
All references cited herein are incorporated herein by reference in their entirety.